Human parainfluenza viruses 1, 2, and 3 (HPIV1, 2, 3) are significant causes of severe pediatric respiratory tract disease worldwide. The HPIVs are enveloped, non-segmented, negative strand RNA viruses of the family Paramyxoviridae. The broad outlines of their biology and molecular genetics have been defined in previous studies by this laboratory and others. The HPIV genome encodes a nucleoprotein N, phosphoprotein P, large polymerase protein L, internal matrix protein M, and fusion F and hemagglutinin-neuraminidase HN transmembrane surface glycoproteins. F and HN are the 2 viral neutralization antigens and the major protective antigens. In addition, the P gene encodes various accessory protein(s) from one or more additional ORFs that have been described in previous years. Presently, our immediate goal is to develop attenuated versions of HPIV1 and 3 that have been engineered to express the fusion F protein of human respiratory syncytial virus (RSV) from an added gene. RSV is the most important viral agent of severe pediatric respiratory tract disease, with a contribution to human disease comparable to or exceeding that of the HPIVs combined, and the RSV F protein is the major RSV neutralization and protective antigen. An HPIV vector expressing the RSV F protein would provide a bivalent vaccine against the respective HPIV and RSV. Compared to RSV strains, the HPIVs replicate more efficiently in cell culture and have much greater physical stability. They also form discrete particles compared to the large filaments of RSV, making them more amenable to filtration and other steps in manufacture. These attributes make HPIV vectors much easier to manufacture, distribute, and use compared to attenuated RSV strains. These advantages may be essential for extending RSV vaccines to resource-challenged countries. Furthermore, in experimental animals, boosting RSV responses to an earlier primary immunization with an attenuated RSV strain was more efficient using an HPIV/RSV vector as opposed to re-dosing with the attenuated RSV strain, since the latter is subject to greater restriction by prior RSV-specific immunity. We have been evaluating a number of parameters of vaccine vector design using, as proof of principle, an attenuated HPIV3 vaccine candidate called B/HPIV3. This virus consists of bovine PIV3 in which the F and HN genes have been replaced by those of HPIV3, yielding a chimeric virus that is attenuated in primates due to the bovine backbone and bears the neutralization and major protective F and HN antigens of HPIV3. Previously, the empty B/HPIV3 vector was evaluated in a phase 1 clinical trial, and was shown to be well-tolerated in infants and young children (NIH study). Subsequently, B/HPIV3 expressing the unmodified RSV F protein (B/HPIV3-RSV-F) was evaluated in a phase 1 clinical trial in infants and young children, and also was well-tolerated (MedImmune study). However, the construct was poorly immunogenic for RSV F, and analysis of nasal wash specimens of shed vaccine showed that 50% of specimens had evidence of loss of expression of RSV F. Therefore, we have been working to increase the immunogenicity and stability of the RSV F insert. We previously evaluated the effects of the position of insertion of the RSV F gene into the B/HPIV3 backbone, and found that the first (pre-N) and second (N-P) gene positions readily accommodated the RSV F insert. This resulted in greatly increased expression of RSV F protein compared to downstream locations (up to 69-fold increase) and greatly increased fusion. Surprisingly, this did not appear to interfere with vector replication in vitro or in hamsters.We also previously found that expression of the RSV F protein was further enhanced 5-fold by codon-optimization and by modifying the amino acid sequence to be identical to that of an early passage of the original clinical isolate. This conferred a hypo-fusogenic phenotype that presumably reflects the original clinical isolate, and suggests that this strain of RSV may have mutated during passage in vitro to acquire a hyper-fusogenic phenotype. We also evaluated a version of pre-fusion RSV F protein (called DS) that was stabilized by an added disulfide bond. In the hamster model, the DS form of RSV F induced increased quantity and quality of RSV-neutralizing serum antibodies and increased protection against wt RSV challenge, compared to native F. We defined high quality antibodies as those that neutralized RSV in vitro in the absence of added complement: remarkably, expression of unmodified F did not induce detectable high quality antibodies in hamsters, whereas pre-F induced a titer of 1:250. During the present report period, we further modified RSV F by replacing its cytoplasmic tail (CT) domain, or its CT plus transmembrane (TM) domains (TMCT), with its counterparts from BPIV3 F. This was done with and without the DS pre-F stabilization. This resulted in RSV F being packaged in the B/HPIV3 particle with an efficiency similar to that of RSV particles. Enhanced packaging was substantially attenuating in hamsters (10- to 100-fold) and rhesus monkeys (100- to 1000-fold). Nonetheless, TMCT-directed packaging substantially increased the titers of high quality serum RSV-neutralizing antibodies in hamsters and rhesus monkeys. In rhesus monkeys, the combination of packaging plus pre-F stabilization resulted in 8- and 30-fold increases of serum RSV-neutralizing titers in the presence and absence of added complement, respectively, compared to pre-F stabilization alone, despite the much-lower replication due to TMCT. The genetic stability of the RSV F insert in the B/HPIV3 vector was evaluated by a double-immunostaining assay designed to detect expression of both RSV F and vector proteins. Surprisingly, the stability of RSV F expression typically was very high and was not affected by enhanced expression, or pre-F stabilization, or packaging via TMCT. An occasional preparation was found to have some loss of expression of RSV F, but this could simply be discarded and replaced by a replicate preparation. These studies provided improved versions of the well-tolerated rB/HPIV3-RSV F vaccine candidate, ones that induce a superior serum RSV-neutralizing antibody response. An improved version can now be returned to clinical trials. As noted above, packaging the RSV F protein into the B/HPV3 vector increased its level of attenuation in rhesus monkeys 100- to 1000-fold, while providing increased quantity and quality of serum RSV-neutralizing antibodies. However, this reduction in replication is not needed for safety, since the B/HPIV3 vector already was satisfactorily attenuated on its own, and the further 100- to 1000-fold reduction in replication may unnecessarily reduce immunogenicity due to reduced antigen expression. Therefore, we also are inserting the TMCT version of RSV F into other backbones that are less restricted than B/HPIV3, and therefore should yield a construct that is not over-restricted for replication and therefore should be more immunogenic. This presently is being evaluated with HPIV3 and HPIV1 as vectors, with and without an added attenuating mutation.